Faculty Bibliography

Cysteine string protein alpha (CSPalpha), a presynaptic cochaperone for Hsc70, is required for synapse maintenance. Deletion of CSPalpha leads to neuronal dysfunction, synapse loss, and neurodegeneration. We utilized unbiased, systematic proteomics to identify putative CSPalpha protein clients. We found 22 such proteins whose levels are selectively decreased in CSPalpha knockout synapses. Of these putative CSPalpha protein clients, two directly bind to the CSPalpha chaperone complex and are bona fide clients. They are the t-SNARE SNAP-25 and the GTPase dynamin 1, which are necessary for synaptic vesicle fusion and fission, respectively. Using hippocampal cultures, we show that CSPalpha regulates the stability of client proteins and synaptic vesicle number. Our analysis of CSPalpha-dynamin 1 interactions reveals unexpectedly that CSPalpha regulates the polymerization of dynamin 1. CSPalpha, therefore, participates in synaptic vesicle endocytosis and may facilitate exo- and endocytic coupling. These findings advance the understanding of how synapses are functionally and structurally maintained.

Cholinergic basal forebrain (CBF) nucleus basalis (NB) neurons display neurofibrillary tangles (NFTs) during Alzheimer's disease (AD) progression, yet the mechanisms underlying this selective vulnerability are currently unclear. Rac1, a member of the Rho family of GTPases, may interact with the proapoptotic pan-neurotrophin receptor p75(NTR) to induce neuronal cytoskeletal abnormalities in AD NB neurons. Herein, we examined the expression of Rac1b, a constitutively active splice variant of Rac1, in NB cholinergic neurons during AD progression. CBF tissues harvested from people who died with a clinical diagnosis of no cognitive impairment (NCI), mild cognitive impairment, or AD were immunolabeled for both p75(NTR) and Rac1b. Rac1b appeared as cytoplasmic diffuse granules, loosely aggregated filaments, or compact spheres in p75(NTR)-positive NB neurons. Although Rac1b colocalized with tau cytoskeletal markers, the percentage of p75(NTR)-immunoreactive neurons expressing Rac1b was significantly increased only in AD compared with both mild cognitive impairment and NCI. Furthermore, single-cell gene expression profiling with custom-designed microarrays showed down-regulation of caveolin 2, GNB4, and lipase A in AD Rac1b-positive/p75(NTR)-labeled NB neurons compared with Rac1b-negative/p75(NTR)-positive perikarya in NCI. These proteins are involved in Rac1 pathway/cell cycle progression and lipid metabolism. These data suggest that Rac1b expression acts as a modulator or transducer of various signaling pathways that lead to NFT formation and membrane dysfunction in a subgroup of CBF NB neurons in AD.

To evaluate molecular signatures of an individual cell type in comparison to the associated region relevant towards understanding the pathogenesis of Alzheimer's disease (AD), CA1 pyramidal neurons and the surrounding hippocampal formation were microaspirated via laser capture microdissection (LCM) from neuropathologically confirmed AD and age-matched control (CTR) subjects as well as from wild type mouse brain using single population RNA amplification methodology coupled with custom-designed microarray analysis with real-time quantitative polymerase-chain reaction (qPCR) validation. CA1 pyramidal neurons predominantly displayed downregulation of classes of transcripts related to synaptic transmission in AD versus CTR. Regional hippocampal dissections displayed downregulation of several overlapping genes found in the CA1 neuronal population related to neuronal expression, as well as upregulation of select transcripts indicative of admixed cell types including glial-associated markers and immediate-early and cell death genes. Gene level distributions observed in CA1 neurons and regional hippocampal dissections in wild type mice paralleled expression mosaics seen in postmortem human tissue. Microarray analysis was validated in qPCR studies using human postmortem brain tissue and CA1 sector and regional hippocampal dissections obtained from a mouse model of AD/Down syndrome (Ts65Dn mice) and normal disomic (2N) littermates. Classes of transcripts that have a greater percentage of the overall hybridization signal intensity within single neurons tended to be genes related to neuronal communication. The converse was also found, as classes of transcripts such as glial-associated markers were under represented in CA1 pyramidal neuron expression profiles relative to regional hippocampal dissections. These observations highlight a dilution effect that is likely to occur in conventional regional microarray and qPCR studies. Thus, single population studies of specific neurons and intrinsic circuits will likely yield informative gene expression profile data that may be subthreshold and/or underrepresented in regional studies with an admixture of cell types

Functional genomics and proteomics approaches are being employed to evaluate gene and encoded protein expression changes with the tacit goal to find novel targets for drug discovery. Genome-wide association studies (GWAS) have attempted to identify valid candidate genes through single nucleotide polymorphism (SNP) analysis. Furthermore, microarray analysis of gene expression in brain regions and discrete cell populations has enabled the simultaneous quantitative assessment of relevant genes. The ability to associate gene expression changes with neuropsychiatric disorders, including bipolar disorder (BP), and their response to therapeutic drugs provides a novel means for pharmacotherapeutic interventions. This review summarizes gene and pathway targets that have been identified in GWAS studies and expression profiling of human postmortem brain in BP, with an emphasis on glutamate receptors (GluRs). Although functional genomic assessment of BP is in its infancy, results to date point towards a dysregulation of GluRs that bear some similarity to schizophrenia (SZ), although the pattern is complex, and likely to be more complementary than overlapping. The importance of single population expression profiling of specific neurons and intrinsic circuits is emphasized, as this approach provides informative gene expression profile data that may be underappreciated in regional studies with admixed neuronal and non-neuronal cell types

The hTau mouse model of tauopathy was utilized to assess gene expression changes in vulnerable hippocampal CA1 neurons. CA1 pyramidal neurons were microaspirated via laser capture microdissection followed by RNA amplification in combination with custom-designed microarray analysis and qPCR validation in hTau mice and nontransgenic (ntg) littermates aged 11-14months. Statistical analysis revealed ~8% of all the genes on the array platform were dysregulated, with notable downregulation of several synaptic-related markers including synaptophysin (Syp), synaptojanin, and synaptobrevin, among others. Downregulation was also observed for select glutamate receptors (GluRs), Psd-95, TrkB, and several protein phosphatase subunits. In contrast, upregulation of tau isoforms and a calpain subunit were found. Microarray assessment of synaptic-related markers in a separate cohort of hTau mice at 7-8months of age indicated only a few alterations compared to the 11-14month cohort, suggesting progressive synaptic dysfunction occurs as tau accumulates in CA1 pyramidal neurons. An assessment of SYP and PSD-95 expression was performed in the hippocampal CA1 sector of hTau and ntg mice via confocal laser scanning microscopy along with hippocampal immunoblot analysis for protein-based validation of selected microarray observations. Results indicate significant decreases in SYP-immunoreactive and PSD-95-immunoreactive puncta as well as downregulation of SYP-immunoreactive and PSD-95-immunoreactive band intensity in hTau mice compared to age-matched ntg littermates. In summary, the high prevalence of downregulation of synaptic-related genes indicates that the moderately aged hTau mouse may be a model of tau-induced synaptodegeneration, and has profound effects on how we perceive progressive tau pathology affecting synaptic transmission in AD

Background: Reductions in brain-derived neurotrophic factor (BDNF) have been implicated in the pathophysiology of depression. Nevertheless, the factors influencing central and peripheral BDNF levels are still poorly understood. Cerebral microvascular endothelial cells are known to be a major source of BDNF within the brain. Exposure of these cells to amyloid beta (Abeta), which may play a role in the pathophysiology of late-life depression, results in cell death or injury with significant reductions in BDNF secretion. Moreover, in rodents, infusion of Abeta40 into the carotid artery resulted in a disruption of endothelial cells, which was not observed with Abeta42 infusion. Therefore, we hypothesized that concentrations of plasma Abeta40, but not Abeta42, would have a negative effect on plasma BDNF levels. Methods: We examined BDNF and Abeta levels in plasma via immunoblotting and ELISA assays, respectively, from 88 subjects with intact cognition (no dementia and a Mini-Mental State Exam score of at least 28) and no gross MRI abnormalities other than white matter hyperintensities. As these subjects were originally recruited for a study on major depressive disorder (MDD), 45 had MDD and 43 were age-matched controls. Results: Consistent with our prediction, Abeta40 levels were inversely correlated with BDNF concentrations (p<.001), whereas Abeta42 levels were independent of BDNF expression (p=.231). This pattern was similar when MDD and control subjects were analyzed separately. Discussion: Our results are consistent with the hypothesis that cerebral endothelial cells are a contributing source of peripheral BDNF and that their disruption by circulating Abeta40 results in reduction in BDNF. However, these preliminary findings need confirmation, and the mechanisms for our observation, including Abeta40-induced cerebral endothelial cell dysfunction, will have to be clarified

Background: Although the hippocampus is selectively vulnerable to neurodegeneration during the early stages of Alzheimer's disease (AD), the molecular and cellular mechanisms underlying this dysfunction are poorly understood. Nerve growth factor, itscognate receptors and downstreamevents aswell as endosomal- lysosomal systemimpairmentmayunderlie hippocampal dysfunction during the progression of AD.Methods: To explore the role of thesemolecular factors in hippocampal degeneration during the progression of AD we performed single population gene expression array analysis and quantitative immunoblotting on tissues obtained from subjects who died with an antemortem clinical diagnosis of nocognitive impairment (NCI), mild cognitive impairment (MCI), or AD. Expression profiling observations were validated with real-time qPCR and immunocytochemistry. Results: In the present investigation, we found an increase in hippocampal protein levels of proNGF (p = 0.027) and to a lesser extent phopho-JNK (Ser 473, p = 0.066) in AD compared to NCI andMCI. Hippocampal p75^NTR remained stable across the three clinical groups while TrkA levels were reduced w60% in MCI compared to NCI orAD. No differences were found in sortilin, NRH2, phospho-AKT, phospho- ERK1/2 orAKT, ERK1/2 and JNK in the hippocampus across groups. ProNGF levels were positively correlated with phospho-JNK and to a lesser extent to phospho-AKT, suggesting activation of downstream cell survival and stress activation signals. Increased proNGF and phospho-JNK levels were associated with lower MMSE scores but not Braak neuropathology. Phospho-AKT and phosphor-ERK1/2levels were not associated with MMSE or Braak stage. On the other hand, single cell expression profiling of hippocampal CA1 neurons indicate a significant upregulation of early endosome effectors rab4 (AD>MCI&NCI) and rab5 (AD>MCI>NCI), late endosome constituent rab7 (AD &MCI > NCI), and the trafficking molecule rab24 (AD > MCI & NCI). Down regulation of thesynaptic-related marker r!